Mino T.⁺*, Adeline S. M. Chua*, Satoh H. and Robert Seviour*

+ : Corresponding author,e-mail:mino@k.u-tokyo.ac.jp

* : Department of Environmental Studies, Graduate School of Frontier Sciences, the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

** : Biotechnology Research Centre, La Trobe University, Bendigo, Australia

Abstract: This study emphasizes the benefits of using techniques like FISH/MAR and PHA staining/MAR to resolve the in situ physiology of the populations of interest. Large, homogenous clusters of coccobacilli were found to be abundant in the biomasses from a conventional plant at Rosebud, Victoria, Australia. Cells with similar feature were also observed in some enhanced biological phosphate removal (EBPR) systems and reported as yeast spores and Rhodocyclus-related polyphosphate accumulating organisms (PAOs). The identity and the in situ physiology of these dominant microorganisms were investigated in this study. Fluorescent in situ hybridization (FISH) probing showed these cells were prokaryotic and members of the b-Proteobacteria. However, these large clustered cells did not respond to the PAO mix FISH probes, although they accumulated polyphosphate. The in situ physiology of these large cells was studied with FISH in combination with microautoradiography (MAR). These cells were able to take up acetate, glutamate and aspartate, but not glucose under both aerobic and anaerobic conditions. Nile Blue A staining in combination with MAR showed cells incubated under anaerobic conditions contained polyhydroxyalkanoates (PHA) granules. In addition, MAR showed aerobic ³³P_i assimilation with all these substrates, consistent with them supporting an EBPR capacity in these large cells.

Keywords: enhanced biological phosphate removal (EBPR), fluorescent in-situ hybridization (FISH), microautoradiography (MAR), polyphosphate accumulating organisms (PAO).

Introduction

Nutrient control in wastewater treatment plant has become essentially important in tackling eutrophication. Phosphorus is considered more crucial than nitrogen (Seviour et al., 2003) as some blue-green bacteria are capable of fixing the atmospheric nitrogen gas for their primary production. Enhanced biological phosphorus removal (EBPR) process has been adopted worldwide in removing P from wastewater. EBPR is achieved by circulating mixture of influent wastewater and returned sludge through an anaerobic stage followed by an aerobic phase (Mino et al., 1998). With this process configuration, particular bacteria known as polyphosphate accumulating organisms (PAOs), capable of accumulating large amounts of phosphorus as polyphosphate (polyP) are enriched. Subsequently, phosphorus removal can be accomplished by wasting P-rich sludge (Mino et al., 1998).

However, since it was put into practice, unreliable performances of EBPR processes have been reported intermittently. This is largely due to little or no real understanding of the microbiology involved (Seviour et al., 2003). The microbial ecology of P removal still remains a great challenge in activated sludge microbiology until now. Thus, many studies have been conducted, in attempts to clarify the microbial ecology of P removal. Acinetobacter species were the first bacteria isolated from biomass with a high P removal capacity (Fuhs and Chen, 1975) and thought to be a putative PAO. However, with the application of molecular RNA-based culture independent methods on community structure of EBPR activated sludge systems, an important role for Acinetobacter spp. as PAO has now been largely discounted in favor of b-Proteobacterial Rhodocyclus related organisms (Wagner & Loy, 2002; Seviour et al., 2003). Apart from that, Actinobacteria and members of a-Proteobacteria (Seviour et al., 2003) are also commonly numerically dominant in EBPR systems. Fluorescence in situ hybridization (FISH) analyses of both full-scale and laboratory scale EBPR communities show these bacteria are often dominant there, suggesting it is unlikely to be one single dominant PAO but several different bacterial groups may be important.

One report has even claimed that not all PAO are bacteria, and the distinctively large clustered cells seen in a full-scale EBPR plant community were in fact yeast spores (Melasniemi et al., 2000). However, the evidence produced to support this suggestion was indirect, and consequently open to criticism, and most surprisingly, no molecular based methods were applied to more convincingly demonstrate the eukaryotic nature of these cells. PAO with a similar morphology and unusual Gram staining reaction (staining neither Gram-positive nor Gram-negative, but characteristically orange brown) have been shown elsewhere (Crocetti et al., 2000) to fluoresce with the FISH probe they designed against the Rhodocyclus related organisms.

Cells with the same feature were abundant in the biomasses from a conventional plant at Rosebud, Victoria, Australia. The aim of this study was to investigate if the large, homogenous clusters of coccobacilli observed were similar to those reported in Melasniemi et al. (2000) and Crocetti et al. (2000), as well as to clarify their in situ physiology. Chemical staining methods such as Gram staining and methylene blue staining were performed in order to understand the basic characteristics of these cells. Their identity was later examined by FISH using 10 oligonucleotide probes, targeting different microbial groups. After that, the in situ physiology of these large cells was studied with FISH/MAR (Lee et al., 1999), in attempts to understand their anaerobic substrate assimilation abilities and subsequent phosphate uptake, to see whether they behaved as expected of PAO and to resolve whether EBPR was possible in the presence of substrates other than acetate, which is commonly used for laboratory scale studies on EBPR (Seviour et al 2003). In addition to short chain volatile fatty acid, amino acids such as glutamate and aspartate, as well as glucose were used as sole carbon sources respectively in the in situ physiological studies. The information of how substrates other than acetate influence EBPR is very essential because full-scale activated sludge systems deal with a much more diverse range of substrates, and yet little is know about it.

Materials and Methods

Sludge samples. The activated sludge samples were taken from the aerobic basin of a municipal wastewater treatment plant in Rosebud, Victoria, Australia. Samples were stored at 4°C and all experiments were performed within 24 hours after sampling. The suspended solids content was between 3-4 g SS/l and was diluted with filtered sludge water to appropriate concentrations for experiments detailed later. Initial screening was with Gram and methylene blue staining, adopted from Lindrea et al. (1999).

Fluorescent staining methods. DAPI-staining (4,6-diamino-2-phenylindoldihydrochloride) was adapted from the protocol of Kawaharasaki et al. (1999) to visualize polyP (330/420). FISH was performed on fixed activated sludge to examine the identity of large clustered cells, according to Manz et al. (1992). The oligonucleotide probe sequences used for FISH in this study are listed in Table 1, attached to the EUBmix probe was the FLOUS fluorochrome (465,515), and all others were labeled with CY3 (540,605).

Table 1. Oligonucleotide probes used in FISH Probe used Sequence (5’-3’) Target bacteria % FA Reference EUBmix338-1 GCTGCCTCCCGTAGGAGT Most bacteria 20-35 Amann et al. (1990) EUB338- II GCAGCCACCCGTAGGTGT Planctomycetes Daims et al. (1999) EUB338-III GCTGCCACCCGTAGGTGT Verrucomicrobiales Daims et al. (1999) ALF968 GGTAAGGTTCTGCGCGTT a-Proteobacteria 20 Neef et al. (1999) BET42a

Competitor GCCTTCCCACTTCGTTT

GCCTTCCCACATCGTTT b-Proteobacteria 35 Manz et al. (1992) GAM42a

Competitor GCCTTCCCACATCGTTT

GCCTTCCCACTTCGTTT g- Proteobacteria 35 Manz et al. (1992) HGC69a TATAGTTACCACCGCCGT Gram positive, high G+C 25 Roller et al. (1994) LGC354A CCGAAGATTCCCTACTGC Low G+C bacteria 35 Meier et al. (1999) CF TGGTCCGTGTCTCAGTAC Cytophaga-Flavobacterium 35 Wagner et al. (1994) PAO462 CCGTCATCTACWCAGGGTATTAAC R. tenuis subgroup 35 Crocetti et al. (2000) PAO846 GTTAGCTACGGCACTAAAAGG R. tenuis subgroup 35 Crocetti et al. (2000) Euk1379 TACAAAGGGCAGGGAC Eukaryotic cells - Hicks et al. (1992)

Substrate uptake determined by Microautoradiography. The general procedures described by Lee et al. (1999 & 2002) were used to study the in situ physiology of the large cells, in attempts to understand their substrate assimilation and phosphate uptake abilities under different conditions. Activated sludge samples (1gSS/l) were incubated under aerobic and anaerobic conditions with [1-¹⁴C]acetate, L-[G-³H]glutamate, L-[2,3-³H]-aspartate, and D-[6-³H]glucose. Samples were preincubated for 1h, followed by a 4h incubation with sterile radioactive and non radioactive substrate. Each reaction mixture had a total volume of 2 ml, with 20mCi of radioactive substrate and a final substrate concentration of 2 mM. To study microbial phosphorus uptake, sludge samples were incubated with ³³P_i under anaerobic:aerobic cycling conditions (Lee et al., 1999). Samples were preincubated under anaerobic conditions for 1hr, then incubated anaerobically with substrates for 2 hours and finally aerobically for 2hrs. Each sample had a total volume of 2ml, contained 15mCi/gSS ³³P_i, with a final P concentration of 0.3mM using monobasic orthophosphate, and 1mM of a non-labeled carbon source (acetate, aspartate, glutamate and glucose). All radioactive chemicals were purchased from Amersham Pharmacia Biotech (Sydney) and all nonlabeled chemicals used were of analytical grade. Parallel with each set of incubations, appropriate controls were performed, as described in Lee et al. (1999).

Sample fixation, washing and storage. The incubations were terminated by adding 8% paraformaldehyde to the sludge samples in 1:1 ratio (Lee et al., 1999, Ito et al., 2002). After fixation for 3h at 4°C, samples were washed three times with tap water in order to remove the excess soluble radioactive substrate and fixative. Samples incubated with ³³P_i, were washed three times in 0.1M Sodium Citrate-HCl buffer (pH2) as described in Lee et al. (1999). Subsequently, samples could then be stored in Ethanol:PBS at 20°C.

FISH/MAR of sludge samples. Smears of MAR samples were prepared on gelatin-coated cover slips and FISH then carried out as described in Lee et al. (1999). All FISH procedures incorporating appropriate controls were performed according to Amann et al (1995). Based on preliminary FISH analyses, the only probes used with MAR were EUBmix (EUB338, EUBII, and EUBIII) probe, BET42a probe, and PAOmix (PAO462, and PAO846) probe (Table 1). After FISH, autoradiographical procedures were performed according to Andreasen and Nielsen (1997). Liquid film emulsion LM1 (Amersham Pharmacia Biotech) and Kodak D19 developer were used. The optimum exposure time for the samples was between 2 and 14 days, determined empirically, depending on the different incubation conditions used and the radioactive substrate.

PHA staining/MAR of sludge samples. In some experiments (see Results), MAR samples for PHA staining were spread onto gelatin-coated cover slips and air dried. Approximately 10-20 ml of 100ppm (w/v) Nile Blue A-ethanol solution was placed on the samples (Kitamura et al., 1994), which were kept in the dark for 15 min. Then samples were washed gently with tap water to remove any excess staining solution and air dried, before autoradiography was performed as described above.

Microscopy. Gram and methylene blue stained samples were examined under a Zeiss (JENAVAL) light microscope. DAPI, FISH, MAR and PHA staining were viewed under a Nikon epifluorescence microscope (Eclipse E800).

Results and Discussion FISH analyses of biomass samples

The large clustered coccobacilli were morphologically very similar to those reported by Melasniemi et al. (2000) and Crocetti et al. (2000), and could be readily seen under phase contrast microscopy. On Gram staining, these cells stained neither typically Gram-positive nor Gram-negative but characteristically orange brown (Fig 1). Crocetti et al. (2000) reported a similar unusual Gram stain reaction with their large clustered cells too. Methylene blue and DAPI staining showed these clustered cells contained polyP granules, suggesting they are possibly PAO candidates (Fig 1). Furthermore, FISH probing showed these cells were clearly prokaryotic, fluorescing strongly with the EUB mix probe of (Daims et al., 1999), and not fluorescing with the eukarya probe of Hicks et al. (1992). The cells were also members of the b-Proteobacteria, responding positively to the BET42A probe (Fig 1). However, these large clustered cells did not respond to the PAO mix FISH probes described by Crocetti et al. (2000) and Zilles et al. (2002) for their Rhodocyclus related organisms. As well as raising doubts about the importance of eukaryotic yeast spores as PAO, these data would suggest that the large cells storing polyP observed here are phylogenetically different to those reported by Crocetti et al. (2000). Smaller clustered cells were also seen in these biomass samples and again contained polyP detectable by staining, and unlike the larger cells described above, fluoresced with the RHC175 FISH probe of Hesselmann et al (1999), but not the PAO mix probes (results not shown).

Fig.1. Large clustered cells. a) Large clustered cells stained orange brown under Gram staining. b) DAPI staining showed these clustered cells contained polyP granules.

FISH/MAR and PHA staining/MAR

The fact that these poly-P-accumulating large clustered cells were neither yeast spores nor the previously described Rhodocyclus related organisms led us to clarifying their in situ physiology with FISH/MAR (Lee et al 1999). As described previously, sludge samples were incubated with several radio-labeled and non-radio-labeled substrates under different conditions, in attempts to clarify what their substrate assimilation and phosphate uptake abilities might be in the presence of various electron acceptors. These cells could take up radio-labeled acetate, glutamate and aspartate, but not glucose under both aerobic and anaerobic conditions (Fig.2, the case of glutamate is shown as example). Anaerobic glutamate and aspartate uptake in EBPR systems was reported previously by Satoh et al. (1998). Our results add further evidence for a possible role for amino acids in supporting EBPR. Nile Blue A staining in combination with MAR showed cells incubated under anaerobic conditions contained poly-hydroxyalkanoates (PHA), a distinctively prokaryotic activity, after incubation with radi-olabeled acetate, glutamate and aspartate, although it was unclear whether these substrates were being used for anaerobic PHA synthesis. Moreover, under aerobic conditions, MAR showed aerobic ³³P_i assimilation with acetate (Fig. 3), glutamate and aspartate, consistent with them supporting an EBPR capacity in these large cells. ³³P_i uptake by mainly Rhodocyclus-related PAOs in EBPR biomass has been reported by Kong et al. (2002) and Lee et al. (2002).

Our results strongly indicate the presence of PAO other than the already implicated Rhodocyclus related organisms in full-scale EBPR systems. The data also suggest that EBPR may be supported by amino acids, signifying the possible role of amino acids in EBPR deserves more attention, as their presence in wastewater is certain (Seviour et al., 2003). While several studies (Jeon et al., 2000, Wang et al., 2002) have convincingly shown that glucose may support stable EBPR in laboratory scale reactors, other reports show EBPR failure with glucose as sole carbon source (Cech et al., 1990, 1993), agreeing with our preliminary data. However, definite conclusions should await a more comprehensive knowledge of EBPR biochemistry, and a better understanding of the structure and function of the responsible microbial communities is gained (Seviour et al., 2003).

As well as raising doubts about a role for yeasts in EBPR, the data suggest that much still needs to be learned about the identity and level of biodiversity of the PAO in EBPR systems, and emphasizes the benefits of using techniques like FISH/MAR and PHA/MAR staining to resolve the in situ physiology of the populations of interest there.

Fig.2. FISH/MAR and PHA staining/MAR. a, b) Aerobic [³H]-glutamate assimilation in large clustered cells fluorescing with the BET42a probe. c, d) Anaerobic [³H]-glutamate assimilation in large clustered cells showing PHA accumulation. The scale bar is 10mm.

Fig.3. Aerobic uptake of ³³P_i by clustered large coccobacilli cells with acetate.

CONCLUSIONS

The clustered large b-Proteobacterial coccobacilli, morphologically similar to those seen by both Melasniemi et al. (2000) and Crocetti et al. (2000) appear to be putative PAO candidate. This study raises doubt about any role for yeast spores in EBPR, and also indicates PAO candidate phylogenetically different from the Rhodocyclus-related PAOs responding to the FISH probes of Crocetti et al (2000) and Zilles et al (2002). These outcomes emphasize how much still needs to be learned about the identity and level of biodiversity of the PAO in EBPR systems. In addition, the application of techniques like FISH/MAR and PHA staining/MAR were shown to be useful to help resolve the in situ physiology of the populations of interest there. Under both aerobic and anaerobic conditions, uptake of radiolabeled acetate, glutamate and aspartate were observed for these large clustered cells. Aerobic ³³P_i assimilation with acetate, glutamate and aspartate as carbon sources was also demonstrated. The large clustered cells seemed not to favor glucose as a carbon source to support EBPR. The combination of PHA staining and MAR suggested a possible conversion of radiolabeled acetate, glutamate and aspartate to intracellular PHA under anaerobic conditions.

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